Project Summary Cell adhesion is crucial aspect of development, fertilization, tissue homeostasis, protection from and interaction with other organisms. Many adhesion proteins have been intensively studied for decades and it was an open question whether we have sufficient knowledge of adhesion, given the large number of interacting proteins and interactions involved in adhesion. LINKIN is a conserved, transmembrane protein that we found to be involved in cell adhesion, suggesting that by studying LINKIN and its interacting proteins we can define a new cellular pathway involved in cell adhesion. The striking conservation of LINKIN sequence and gene copy number across eukaryotic evolution suggests a critical role in development. We discovered LINKIN's role in cell adhesion in the context of C. elegans development, specifically the attachment of the migrating linker cell to the vas deferens, which provides a powerful assay for its structure, function and interactions. We identified interacting proteins in human cells and provided evidence for similar molecular interactions in both organisms. We identified the RUVBL1 and RUVBL2 AAA-ATPases, and ?-tubulin as cytoplasmic interactors of LINKIN, suggesting a role for LINKIN in regulating microtubule dynamics, but further investigation is required to connect LINKIN to other interactors and signaling pathways. We propose to further characterize LINKIN's role in cell adhesion in order to connect this new adhesion molecule to other known (or unknown) cellular pathways. A major limitation of human genetic, genomic and systems biology approach is our understanding of the function of many genes. By discovering a new pathway, we will increase the likelihood that genomic, clinical and genetic studies will be interpretable. We will analyze the function and interactors of both the extracellular and intracellular domains of LINKIN. Using evolutionary conservation as a guide we will mutate conserved residues in C. elegans LINKIN and test their function in transgenic worms. We will use human LINKIN proteins with cognate mutations in cell lines for immunoprecipitation and quantitative mass spectrometry proteomics to identify physiologically relevant interacting proteins. We will test the function of interacting proteins identified by the proteomics in C. elegans and cell lines. We will also use C. elegans genetics to screen prioritized candidates for a role in adhesion using the linker cell attachment assay. Candidates will be prioritized by expression in the linker cell, for which we have a deep transcriptomic profile, predictions of encoding transmembrane or secreted proteins, phylogenetic co-occurrence as well as network predictions of interactions based on orthologous proteins in other intensively-studied organisms. In this way, we will identify the portions of LINKIN that are crucial to its function in adhesion and physiologically-relevant interacting proteins both on other cells and same cell that likely mediate adhesion, and in the cytoplasm that implement the adhesive and cytoskeletal interactions. This validated set of functionally interacting proteins will define a new pathway of cellular adhesion and inform a variety of human genetic studies in normal development and disease states.